Bottom Line:
It was found that Na,K-ATPase α-subunit has a basal glutathionylation which is not abrogated by reducing agent.We have analyzed all X-ray structures of the Na,K-ATPase α-subunit from pig kidney and found that there are a number of isolated cavities with unresolved electron density close to the relevant cysteine residues.Here, we discuss the role of basal glutathionylation of Na,K-ATPase α-subunit and provide evidence supporting the view that this modification is cotranslational.

ABSTRACTMany viruses induce oxidative stress and cause S-glutathionylation of Cys residues of the host and viral proteins. Changes in cell functioning during viral infection may be associated with glutathionylation of a number of key proteins including Na,K-ATPase which creates a gradient of sodium and potassium ions. It was found that Na,K-ATPase α-subunit has a basal glutathionylation which is not abrogated by reducing agent. We have shown that acute hypoxia leads to increase of total glutathionylation level of Na,K-ATPase α-subunit; however, basal glutathionylation of α-subunit increases under prolonged hypoxia only. The role of basal glutathionylation in Na,K-ATPase function remains unclear. Understanding significance of basal glutathionylation is complicated by the fact that there are no X-ray structures of Na,K-ATPase with the identified glutathione molecules. We have analyzed all X-ray structures of the Na,K-ATPase α-subunit from pig kidney and found that there are a number of isolated cavities with unresolved electron density close to the relevant cysteine residues. Analysis of the structures showed that this unresolved density in the structure can be occupied by glutathione associated with cysteine residues. Here, we discuss the role of basal glutathionylation of Na,K-ATPase α-subunit and provide evidence supporting the view that this modification is cotranslational.

fig6: S-glutathionylation of α1-subunit of Na,K-ATPase after acute and prolonged hypoxia before and after TCEP (25 mM) treatment. SC-1 cells were grown either for 3.5 h ((a), (b)) or for 96 h (c), each at 20% and 0.2% pO2. Cell lysates were incubated with ((b), (c)) or without (a) 25 mM TCEP during 30 min at 37°C and α1-subunit of Na,K-ATPase was detected by immunoblotting (IB). (d) α1-Subunit of Na,K-ATPase was immunoprecipitated (IP) from cell lysates by anti-α1 antibodies and glutathionylation was detected with anti-glutathione (anti-GS) antibodies. The original immunoblotting readouts are presented above. Bars represent changes in the S-glutathionylated (GSS-α/α) form of the protein normalized to its total amount. n = 3, mean ± SD. ((e), (f)) Immunoblots of Na,K-ATPase α1-subunit after immunoprecipitation with anti-α1 antibodies and TCEP (25 mM) treatment in denaturing conditions (8 M urea, 8% SDS). (e) Detection of total α1-subunit using anti-α1 antibodies. (f) Detection of glutathionylated α1-subunit using anti-GS antibodies.

Mentions:
It can be hypothesized that the basal glutathionylation depends on the redox status of cells during protein folding. To test this, we incubated SC-1 cells at 0.2% and 20% pO2 within 3.5 h and 96 h. Then, cell lysates were treated with the reducing agent TCEP (25 mM) for 30 min. Acute hypoxia (3.5 h) leads to increase of total glutathionylation level of Na,K-ATPase α-subunit (Figure 6(a)) that corresponds to our data, obtained earlier [13, 28]. However, basal glutathionylation of α-subunit does not change at these conditions (Figure 6(b)). In contrast, at prolonged hypoxia (96 h), basal level of Na,K-ATPase α-subunit glutathionylation was significantly higher than that under 20% pO2 (Figure 6(c)). Since the basal glutathionylation is not removed by reducing agents and the glutathionylated cysteines are located in isolated cavities, we assume that the reaction of glutathionylation occurs during protein folding. If so, then the basal glutathionylation can be removed only from the unfolded protein. We performed immunoprecipitation of Na,K-ATPase α-subunit from lysates of SC-1 cells incubated at 0.2% and 20% pO2 within 3.5 h and 96 h. Analysis of immunoprecipitated α-subunit revealed increasing of its glutathionylation under hypoxic conditions (Figure 6(d)). Then, we treated Na,K-ATPase α-subunit obtained by immunoprecipitation with TCEP under denaturing conditions (8 M urea and 8% SDS). In this case, the glutathionylation was fully removed (Figures 6(e) and 6(f)). Based on these data, we can conclude that the basal glutathionylation is a cotranslational modification which, for example, is necessary to prevent the formation of disulphide bridges between the neighboring cysteine residues during protein folding. Formation of disulfide bridges can increase rigidity of the structure and prevent conformational lability of the molecule. In particular, residues Cys 204 and Cys 242 are located in the actuator domain of Na,K-ATPase that performs large amplitude transitions during the catalytic cycle, which would be impossible if SS bridges were formed between these residues. In addition, the formation of SS bridge between Cys 204 and Cys 242 residues will prevent the exposition of SH group of Cys 242 to the solvent from the cavity and its regulatory glutathionylation. It is also possible that glutathionylation is necessary for correct protein folding. Only glutathione and oxidized glutathione are required for glutathionylation of α-subunit of Na,K-ATPase [13] without any additional enzymes and cofactors. Thus, the ability of SH group to undergo glutathionylation directly depends on the redox state of cells [5]. Reduced SH groups interact with GSSG, the level of which increases in oxidative stress. In the case of oxidation of thiol groups to SOН, they interact with GSH, the concentration of which in cytosol is 1–5 mM.

fig6: S-glutathionylation of α1-subunit of Na,K-ATPase after acute and prolonged hypoxia before and after TCEP (25 mM) treatment. SC-1 cells were grown either for 3.5 h ((a), (b)) or for 96 h (c), each at 20% and 0.2% pO2. Cell lysates were incubated with ((b), (c)) or without (a) 25 mM TCEP during 30 min at 37°C and α1-subunit of Na,K-ATPase was detected by immunoblotting (IB). (d) α1-Subunit of Na,K-ATPase was immunoprecipitated (IP) from cell lysates by anti-α1 antibodies and glutathionylation was detected with anti-glutathione (anti-GS) antibodies. The original immunoblotting readouts are presented above. Bars represent changes in the S-glutathionylated (GSS-α/α) form of the protein normalized to its total amount. n = 3, mean ± SD. ((e), (f)) Immunoblots of Na,K-ATPase α1-subunit after immunoprecipitation with anti-α1 antibodies and TCEP (25 mM) treatment in denaturing conditions (8 M urea, 8% SDS). (e) Detection of total α1-subunit using anti-α1 antibodies. (f) Detection of glutathionylated α1-subunit using anti-GS antibodies.

Mentions:
It can be hypothesized that the basal glutathionylation depends on the redox status of cells during protein folding. To test this, we incubated SC-1 cells at 0.2% and 20% pO2 within 3.5 h and 96 h. Then, cell lysates were treated with the reducing agent TCEP (25 mM) for 30 min. Acute hypoxia (3.5 h) leads to increase of total glutathionylation level of Na,K-ATPase α-subunit (Figure 6(a)) that corresponds to our data, obtained earlier [13, 28]. However, basal glutathionylation of α-subunit does not change at these conditions (Figure 6(b)). In contrast, at prolonged hypoxia (96 h), basal level of Na,K-ATPase α-subunit glutathionylation was significantly higher than that under 20% pO2 (Figure 6(c)). Since the basal glutathionylation is not removed by reducing agents and the glutathionylated cysteines are located in isolated cavities, we assume that the reaction of glutathionylation occurs during protein folding. If so, then the basal glutathionylation can be removed only from the unfolded protein. We performed immunoprecipitation of Na,K-ATPase α-subunit from lysates of SC-1 cells incubated at 0.2% and 20% pO2 within 3.5 h and 96 h. Analysis of immunoprecipitated α-subunit revealed increasing of its glutathionylation under hypoxic conditions (Figure 6(d)). Then, we treated Na,K-ATPase α-subunit obtained by immunoprecipitation with TCEP under denaturing conditions (8 M urea and 8% SDS). In this case, the glutathionylation was fully removed (Figures 6(e) and 6(f)). Based on these data, we can conclude that the basal glutathionylation is a cotranslational modification which, for example, is necessary to prevent the formation of disulphide bridges between the neighboring cysteine residues during protein folding. Formation of disulfide bridges can increase rigidity of the structure and prevent conformational lability of the molecule. In particular, residues Cys 204 and Cys 242 are located in the actuator domain of Na,K-ATPase that performs large amplitude transitions during the catalytic cycle, which would be impossible if SS bridges were formed between these residues. In addition, the formation of SS bridge between Cys 204 and Cys 242 residues will prevent the exposition of SH group of Cys 242 to the solvent from the cavity and its regulatory glutathionylation. It is also possible that glutathionylation is necessary for correct protein folding. Only glutathione and oxidized glutathione are required for glutathionylation of α-subunit of Na,K-ATPase [13] without any additional enzymes and cofactors. Thus, the ability of SH group to undergo glutathionylation directly depends on the redox state of cells [5]. Reduced SH groups interact with GSSG, the level of which increases in oxidative stress. In the case of oxidation of thiol groups to SOН, they interact with GSH, the concentration of which in cytosol is 1–5 mM.

Bottom Line:
It was found that Na,K-ATPase α-subunit has a basal glutathionylation which is not abrogated by reducing agent.We have analyzed all X-ray structures of the Na,K-ATPase α-subunit from pig kidney and found that there are a number of isolated cavities with unresolved electron density close to the relevant cysteine residues.Here, we discuss the role of basal glutathionylation of Na,K-ATPase α-subunit and provide evidence supporting the view that this modification is cotranslational.

ABSTRACTMany viruses induce oxidative stress and cause S-glutathionylation of Cys residues of the host and viral proteins. Changes in cell functioning during viral infection may be associated with glutathionylation of a number of key proteins including Na,K-ATPase which creates a gradient of sodium and potassium ions. It was found that Na,K-ATPase α-subunit has a basal glutathionylation which is not abrogated by reducing agent. We have shown that acute hypoxia leads to increase of total glutathionylation level of Na,K-ATPase α-subunit; however, basal glutathionylation of α-subunit increases under prolonged hypoxia only. The role of basal glutathionylation in Na,K-ATPase function remains unclear. Understanding significance of basal glutathionylation is complicated by the fact that there are no X-ray structures of Na,K-ATPase with the identified glutathione molecules. We have analyzed all X-ray structures of the Na,K-ATPase α-subunit from pig kidney and found that there are a number of isolated cavities with unresolved electron density close to the relevant cysteine residues. Analysis of the structures showed that this unresolved density in the structure can be occupied by glutathione associated with cysteine residues. Here, we discuss the role of basal glutathionylation of Na,K-ATPase α-subunit and provide evidence supporting the view that this modification is cotranslational.